The opening of the 21st century marked the beginning of a fundamental transformation in drug discovery made feasible by advances in genomics technologies. RNA interference now allows the identification of genes central to human disease processes through genome-wide loss-of-function screens in mammalian cells. Similarly, chemical genetic screens in mammalian cells can provide small molecules that are valuable research tools and drug leads. However, although each screening approach generates its own set of biologically important hits, the results of both types of screen are usually evaluated serially and in isolation. This approach fails to identify common pathways targeted by both types of screens. We propose to develop the experimental and analytic tools needed to identify common pathways targeted by hits from both chemical and genetic screens using high-throughput gene expression profiling. We have developed a gene expression-based high-throughput screening (GE-HTS) platform and applied it to chemical screens in primary human T cells. Expression profiling provides a common language to characterize the molecular phenotypes not only of chemical but also of genetic manipulations. We will use GE-HTS to profile hits from parallel small-molecule and RNAi screens, and generate a compendium of signatures caused by chemical and genetic perturbations in primary human T cells. The compendium will then be mined for compounds or genes that induce similar gene expression signatures that would indicate a common target biologic pathway. 1) Identify genes essential for PD-1 inhibition using genome-scale RNA interference Hypothesis: Genes can be identified that are essential for PD-1-mediated inhibition but dispensable for J cell activation. 2) Discover small molecules that relieve PD-1 inhibition in primary human T cells using highthroughput chemical screening Hypothesis: The PD-1 signaling pathway contains proteins that can be selectively targeted by small molecules to reverse T cell inhibition. 3) Integrate chemical and genetic perturbations using high-throughput gene expression profiling to identify common biological target pathways Hypothesis: Small molecule inhibitors of a specific pathway will induce similar transcriptional signatures as genetic disruption of the same pathway.